The long-term objective of the program project is to elucidate the genetic features responsible for the exceptional virulence of the 1918 'Spanish' influenza pandemic. In ongoing work, we have been able to generate sequences of the open reading frames of five of the virus' eight gene segments using fixed and frozen autopsy tissue of 1918 influenza victims. In the current proposal we plan to extend our sequence analyses to the non-coding ends of the 1918 viral gene segments, and to develop techniques to enrich both for influenza RNA and host mRNA from circa 1918 influenza tissue samples. The following specific aims are therefore proposed: Aim #1. Obtain the non-coding sequences of the eight 1918 influenza virus gene segments so that complete 1918 gene segments can be rescued in the proposed collaborative studies of the program project. We propose to obtain the sequences of the non-coding ends of the viral segments by 5'- and 3'-rapid amplification of cDNA ends. It is possible that sequence differences in the promoter regions of the non-coding ends contributed to the unique pathophysiology of the 1918 influenza virus. Aim #2. Develop techniques to make influenza RNA fragment enriched cDNA libraries from circa 1918 fixed tissue samples. Should RT-PCR positive influenza pre-1918 cases be found, the amount of RNA that can be extracted individual blocks will be inadequate for detailed genetic analyses. We propose to produce segment-specific cDNA libraries that could be sequenced using shotgun-sequencing techniques. Aim #3. Develop techniques to amplify host mRNA and produce tissue-based cDNA libraries from circa 1918 tissue samples sufficient in quality and quantity to allow for gene expression analyses with oligonucleotide arrays or by quantitative gene-specific RT-PCR. This will allow direct correlation of gene expression signatures of 1918 influenza virulence obtained with tissue culture or animal models with the expression patterns in the primary human 1918 lung tissues.